Thiophosphorylation of the 130-kDa Subunit Is Associated with a Decreased Activity of Myosin Light Chain Phosphatase in α-Toxin-permeabilized Smooth Muscle

Trinkle-Mulcahy, Laura; Ichikawa, Kazuhito; Hartshorne, David J.; Siegman, Marion J.; Butler, Thomas M.

doi:10.1074/jbc.270.31.18191

Cited by 100 publications

(104 citation statements)

References 28 publications

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“…Inhibition of MLCP by rho also was shown with cultured vascular SMCs (Noda et al, 1995). Phosphorylation of M130 was shown to inhibit phosphatase activity in skinned muscle (Trinkle-Mulcahy et al, 1995). The activity of MLCP also is regulated by phosphorylation of a threonine residue in M130 in vitro (Ichikawa et al, 1996b).…”

Section: Localization Of Mlcp In Migrating Fibroblastsmentioning

confidence: 96%

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Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Murata

Hirano

Villa-Moruzzi

et al. 1997

MBoC

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Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the 8 isoform of type 1 catalytic subunit (PP1C6), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PPlCa isoform colocalized with M130 and also was in the nucleus, whereas the PP1C6 isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.

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Section: Localization Of Mlcp In Migrating Fibroblastsmentioning

confidence: 96%

“…Monoclonal antibody for M130 was made as described before (Trinkle-Mulcahy et al, 1995). Rabbit polyclonal antibody against M130 was made against a recombinant protein encoding 1-674 of M130 (Ichikawa et al, 1996b) and purified through DEAE Affi-gel blue (Bio-Rad, Hercules, CA).…”

Section: Antibody Reagents and Western Blotting Analysismentioning

confidence: 99%

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Murata

Hirano

Villa-Moruzzi

et al. 1997

MBoC

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“…Calcium-activated MLC kinase or Rho-associated kinase (' ROCK '), which are activated by the small GTPase Rho, phosphorylate MYPT1\2, which leads to the inhibition of PP1 activity toward MLC [17,[61][62][63]. The major protein phosphatase that dephosphorylates myosin has been described as a heterotrimer containing the PP1 catalytic subunit, an approx.…”

Section: Discussionmentioning

confidence: 99%

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

Skinner

Saltiel

2001

Biochemical Journal

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To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.

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“…One such mechanism is via phosphorylation of MYPT1 at an inhibitory site, ie, T695 in the larger chicken gizzard isoform. 29 This originated with the observation of Trinkle-Mulcahy et al 31 that incubation of permeabilized rabbit portal vein strips with ATP␥S caused thiophosphorylation of MYPT1 and inhibition of MP activity. Subsequently, it was found that a kinase(s) that co-purified with the MP preparations also phosphorylated MYPT1 at T695 and inhibited phosphatase activity.…”

Section: Shin Et Al Myosin Phosphatase Subunit Localization 551mentioning

confidence: 99%

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

Shin

Gallant

et al. 2002

Circulation Research

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Abstract-It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca 2ϩ . One mechanism that has been suggested to explain Ca 2ϩ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase. (Circ Res. 2002;90:546-553.)

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Thiophosphorylation of the 130-kDa Subunit Is Associated with a Decreased Activity of Myosin Light Chain Phosphatase in α-Toxin-permeabilized Smooth Muscle

Cited by 100 publications

References 28 publications

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

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